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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2012 - April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
adopted 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5-8 weekds
- Weight at study initiation: on average 27.9g
- Assigned to test groups randomly: yes
- Housing: single in Makrolon type M II cages
- Diet (e.g. ad libitum): standardized pelleted feed ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO (4mL/kg b.w.) + corn oil
- Justification for choice of solvent/vehicle: solubility of the test substance, historic control data is available
- Concentration of test material in vehicle: 25 - 100mg/mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved by thourough shaking in DMSO (4 mL/kg) and than emulsified in corn oil (up to 10 mL/kg). All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
one admistration
Frequency of treatment:
one admistration
Post exposure period:
24h , 48h (only high dose)
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP), Vincristine sulfate (VCR)
- Justification for choice of positive control(s): both substances are well-established reference clastogens and aneugens, respectively
- Route of administration: via gavage in deionized water
- Doses / concentrations: 20mg/kg (CPP), 0.15mg/kg (VCR)

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest to determine the acute oral toxicity in males and females, lethality was observed at the recommended highest dose of 2 000 mg/kg body weight. In addition, clear signs of toxicity were observed: piloerection, hunched posture and reduced general condition. However, there were no distinct differences in clinical observations between male and female animals. Thus, only male animals were used in the main experiment. Based on the data of the pretest a dose of 1 000 mg/kg body weight was defined as MTD (maximum tolerated dose) and was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
One drop of isolated bone marrow cells in FCS was dropped onto a clean microscopic slide, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained with eosin and methylene blue (modified May-Grünwald solution) for about 5 minutes. After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes. Subsequently, the slides were stained with Giemsa solution for about 15 minutes. After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Fraction of polychromatic erythrocytes containing micronuclei (index of clastogenic or aneugenic activity)
• Fraction of normochromatic erythrocytes containing micronuclei (24h value: control value for situation before test substance administration)
• Ratio of polychromatic to normochromatic erythrocytes (indicator that the test substance reached the bone marrow)
• Number of small micronuclei (d < D/4) and of large micronuclei (d = D/4) [d = diameter of micronucleus, D = cell diameter] (differentiation between a clastogenic and spindle poison effect, respectively)
Evaluation criteria:
Acceptance criteria
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. = 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

A finding is considered positive, if the number of PCEs containing micronuclei is statistically significant and dose-related increased, and the number of PCEs containing micronuclei exceeds both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance is considered negative, if the number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
PCE to NCE ratio suggests a slight inhibition of erythropoiesis at 1000mg/kg after 48h
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Vehicle control male mice showed frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes as indication of test substance toxicity in the target organ was detected at 48-hour sacrifice interval. No increase in the number of polychromatic erythrocytes containing either small or large micronuclei was detected after test substance administration.

Applicant's summary and conclusion

Conclusions:
Under the condition of this study, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.
Executive summary:

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to 5 male animals per group at dose levels of 250 mg/kg, 500 mg/kg and 1 000 mg/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours (all dosages and control), and 48 hours (high dose and vehicle control) after administration. The preparations were stained, and 2 000 polychromatic erythrocytes were evaluated per animal. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

Vehicle control male mice showed frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes as indication of test substance toxicity in the target organ was detected at 48-hour sacrifice interval. No increase in the number of polychromatic erythrocytes containing either small or large micronuclei was detected after test substance administration. Thus, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.